CN111849930B - Mi-2重组抗原及其制备方法和应用 - Google Patents
Mi-2重组抗原及其制备方法和应用 Download PDFInfo
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- CN111849930B CN111849930B CN202010835800.0A CN202010835800A CN111849930B CN 111849930 B CN111849930 B CN 111849930B CN 202010835800 A CN202010835800 A CN 202010835800A CN 111849930 B CN111849930 B CN 111849930B
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Abstract
本发明涉及生物技术领域,具体而言,提供了一种Mi‑2重组抗原及其制备方法和应用。本发明提供一种Mi‑2重组抗原,包括SEQ ID NO.1、SEQ ID NO.5或SEQ ID NO.7中的至少一种,并且,不含有SEQ ID NO.2和/或SEQ ID NO.6。该抗原提高了Mi‑2抗体检测的灵敏度和特异性,用于Mi‑2抗体检测或多发性肌炎诊断,可以大大提高准确性,为生产应用提供新的技术支持。
Description
技术领域
本发明涉及生物技术领域,具体而言,涉及一种Mi-2重组抗原及其制备方法和应用。
背景技术
多发性肌炎(polymyositis,PM)是一种常见的自身免疫性疾病,Mi-2抗体是肌炎特异性抗体的一种,对肌炎的诊断具有重要意义。抗Mi-2抗体的靶抗原是位于细胞核核质内相对分子质量218kD的Mi-2抗原蛋白。
获取天然的Mi-2抗原常用的方法是从兔胸腺中提取,提取的难度较高且产量很低。Mi-2抗原蛋白由1,912个氨基酸构成,蛋白相对分子量较大,而使用昆虫细胞重组表达的全长Mi-2抗原蛋白,虽容易获得,但免疫原性较低,不能满足诊断试剂的使用性能。
有鉴于此,特提出本发明。
发明内容
本发明的第一目的在于提供一种Mi-2重组抗原。
本发明的第二目的在于提供上述Mi-2重组抗原相关的生物材料。
本发明的第三目的在于提供上述Mi-2重组抗原的制备方法。
本发明的第四目的在于提供上述Mi-2重组抗原的应用。
本发明的第五目的在于提供一种检测多发性肌炎的产品。
为了实现本发明的上述目的,特采用以下技术方案:
一种Mi-2重组抗原,所述Mi-2重组抗原包括SEQ ID NO.1、SEQ ID NO.5或SEQ IDNO.7中的至少一种,并且,不含有SEQ ID NO.2和/或SEQ ID NO.6。
进一步地,所述Mi-2重组抗原还不含有SEQ ID NO.3、SEQ ID NO.4或SEQ ID NO.8中的至少一种。
进一步地,所述Mi-2重组抗原包括(a)-(d)的任一种:
(a)SEQ ID NO.1和SEQ ID NO.5;
(b)SEQ ID NO.1和SEQ ID NO.7;
(c)SEQ ID NO.5和SEQ ID NO.7;
(d)SEQ ID NO.1、SEQ ID NO.5和SEQ ID NO.7;
并且,(a)-(d)任一种中的序列之间通过连接肽连接;
优选地,所述连接肽为GGGGSGGGG。
进一步地,所述Mi-2重组抗原为SEQ ID NO.9或SEQ ID NO.10中的任一种。
与上述Mi-2重组抗原相关的生物材料,所述生物材料为如下任一种:
(a)核酸分子,编码上述Mi-2重组抗原;
(b)表达盒,含有(a)中核酸分子;
(c)重组载体,含有(a)中核酸分子或(b)中表达盒;
(d)重组真核细胞,含有(a)中核酸分子、(b)中表达盒或(c)中重组载体;
(e)重组原核细胞,含有(a)中核酸分子、(b)中表达盒或(c)中重组载体。
Mi-2重组抗原的制备方法,将编码Mi-2重组抗原的核酸分子导入宿主获得重组表达***,经表达得到Mi-2重组抗原。
进一步地,编码Mi-2重组抗原的核酸分子与pET-28a载体构建重组载体,将重组载体导入大肠杆菌,经表达得到Mi-2重组抗原。
Mi-2重组抗原在检测Mi-2抗体或制备检测Mi-2抗体的产品中的应用。
Mi-2重组抗原在制备检测多发性肌炎的产品中的应用。
一种检测多发性肌炎的产品,所述产品采用上述Mi-2重组抗原作为检测抗原。
与现有技术相比,本发明的有益效果为:
本发明提供一种Mi-2重组抗原,该抗原在Mi-2抗原蛋白(UniProtKB-Q14839(CHD4_HUMAN))的基础上进行优化得到,克服了现有技术中天然Mi-2抗原蛋白提取难度高、产量低、成本高的问题,同时也克服了重组全长Mi-2抗原蛋白免疫原性低的缺陷。该抗原显著改善了Mi-2抗体检测的空间位阻问题,抗原位点得到更加充分暴露,提高了Mi-2抗体检测的灵敏度和特异性。将本发明的抗原用于Mi-2抗体检测或多发性肌炎诊断,可以大大提高准确性,为生产应用提供新的技术支持。
附图说明
图1为本发明实施例中融合蛋白NCM的SDS-PAGE检测结果。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述,但是本领域技术人员将会理解,下列实施例仅用于说明本发明,而不应视为限制本发明的范围。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。
除非另有说明,本文中所用的专业与科学术语与本领域熟练人员所熟悉的意义相同。此外,任何与所记载内容相似或均等的方法或材料也可应用于本发明中。
本发明提供一种Mi-2(抗组蛋白乙酰转移酶复合物解旋酶成分)重组抗原,包括SEQ ID NO.1、SEQ ID NO.5或SEQ ID NO.7中的至少一种,并且,不含有SEQ ID NO.2和/或SEQ ID NO.6。
上述抗原在Mi-2抗原蛋白(UniProtKB-Q14839(CHD4_HUMAN))的基础上进行优化得到,克服了现有技术中天然Mi-2抗原蛋白提取难度高、产量低、成本高的问题,同时也克服了重组全长Mi-2抗原蛋白免疫原性低的缺陷。该抗原显著改善了Mi-2抗体检测的空间位阻问题,抗原位点得到更加充分暴露,提高了Mi-2抗体检测的灵敏度和特异性。发明人在试验中发现,相较于天然Mi-2抗原蛋白,本发明的Mi-2重组抗原的检测灵敏度可以提高3倍以上,特异性可以提高1.5倍以上,效果显著。
在本发明中,“Mi-2抗体”指可以与Mi-2蛋白或多肽产生“抗原-抗体”特异性结合的蛋白、多肽或其他物质,也可称为“抗Mi-2抗体”;“Mi-2抗原”或“Mi-2重组抗原”指可以与Mi-2抗体产生“抗原-抗体”特异性结构的蛋白或多肽。
需要说明的是,当Mi-2重组抗原为含有两种或三种序列号氨基酸序列情况时,其构建的融合蛋白可以为含有SEQ ID NO.1和SEQ ID NO.5,SEQ ID NO.1和SEQ ID NO.7,SEQID NO.5和SEQ ID NO.7;或者,SEQ ID NO.1、SEQ ID NO.5和SEQ ID NO.7。其中,任意一种融合蛋白的序列号氨基酸序列之间的顺序并不作特殊限定,例如含有SEQ ID NO.1和SEQID NO.5的融合蛋白上,可以依次含有SEQ ID NO.1和SEQ ID NO.5,也可以依次含有SEQ IDNO.5和SEQ ID NO.1。
在优选地实施方式中,Mi-2重组抗原还不含有SEQ ID NO.3、SEQ ID NO.4或SEQID NO.8中的至少一种。
在优选的实施方式中,为了减少空间位阻同时充分暴露抗原位点,并且缩短抗原序列长度,当Mi-2重组抗原含有两种或三种序列号氨基酸序列时,其构建的融合蛋白之间通过连接肽连接。例如,融合蛋白可以为如下结构:SEQ ID NO.1-连接肽-SEQ ID NO.5、SEQID NO.5-连接肽-SEQ ID NO.1、SEQ ID NO.1-连接肽-SEQ ID NO.5-连接肽-SEQ ID NO.7、SEQ ID NO.5-连接肽-SEQ ID NO.1-连接肽-SEQ ID NO.7、SEQ ID NO.1-连接肽-SEQ IDNO.7-连接肽-SEQ ID NO.5、或者,SEQ ID NO.7-连接肽-SEQ ID NO.1-连接肽-SEQ IDNO.5等等,其中,含有三段序列号氨基酸序列的融合蛋白中,两个“连接肽”可以相同,也可以不同。
在更优选的实施方式中,连接肽可以为GGGGSGGGG。
本发明同时保护氨基酸序列如SEQ ID NO.9或SEQ ID NO.10任一种的Mi-2重组抗原。
本发明还保护与上述Mi-2重组抗原相关的生物材料,例如核酸分子、表达盒、重组载体、重组真核细胞和重组原核细胞等等。可以理解的是上述生物材料均为编码或表达Mi-2重组抗原的生物材料,可以作为生物模块直接用于分子生物学的操作中。
“核酸分子”可以是DNA,如cDNA等,也可以是RNA,如mRNA等。
“表达盒”包含多核苷酸序列,多核苷酸序列编码待表达的Mi-2重组抗原和控制其表达的序列如启动子以及任选地增强子序列,包括顺式作用转录性控制单元的任何组合。控制基因表达(即其转录和转录产物翻译)的序列常称作调节单元。调节单元的大多数部分位于基因的编码序列上游并与之有效连接。表达盒也可以含有包含多聚腺苷化位点的下游3'非翻译区。
“重组载体”的载体可以为质粒、噬菌体或病毒。
“重组真核细胞”的宿主细胞可以为酵母、哺乳动物细胞、昆虫细胞等。
“重组原核细胞”的宿主细胞可以为细菌等。
本发明还保护上述Mi-2重组抗原的制备方法,将编码Mi-2重组抗原的核酸分子导入宿主获得重组表达***,经表达得到Mi-2重组抗原。该方法极大降低了Mi-2重组抗原的生产成本,可以大量获得Mi-2重组抗原,表达***相对成熟,操作简便。优选地,编码Mi-2重组抗原的核酸分子与pET-28a载体构建重组载体,将重组载体导入大肠杆菌,经表达得到Mi-2重组抗原。
将本发明的Mi-2重组抗原用于Mi-2抗体检测或多发性肌炎诊断,可以大大提高准确性,为生产应用提供新的技术支持。
本发明最后保护采用Mi-2重组抗原作为检测抗原的多发性肌炎诊断或Mi-2抗体检测的产品。
下面通过具体的实施例进一步说明本发明,但是,应当理解为,这些实施例仅仅是用于更详细地说明之用,而不应理解为用于以任何形式限制本发明。
实施例1
通过对Mi-2抗原蛋白(UniProtKB-Q14839(CHD4_HUMAN),1912aa)的抗原表位进行生物信息学分析,把Mi-2分成8段分别在大肠杆菌中表达。分别对应的氨基酸序列SEQ IDNO.1(amino 1-239),SEQ ID NO.2(amino 240-448),SEQ ID NO.3(amino 449-677),SEQID NO.4(amino 678-948),SEQ ID NO.5(amino 949-1209),SEQ ID NO.6(amino 1210-1498),SEQ ID NO.7(amino 1499-1696)和SEQ ID NO.8(amino 1697-1912)。
质粒构建与蛋白表达
【1】8个片段对应的引物如下:
SEQ ID NO.1片段的引物为:
F1:5’-atggcgtcgggcctgggc-3’(SEQ ID NO.11);
R1:5’-gctctccaccacagctac-3’(SEQ ID NO.12);
SEQ ID NO.2片段的引物为:
F2:5’-atggtgacagccactgaggttg-3’(SEQ ID NO.13);
R2:5’-atggtggtcatcctcctcttcg-3’(SEQ ID NO.14);
SEQ ID NO.3片段的引物为:
F3:5’-atggaattctgtcgggtctgcaagg-3’(SEQ ID NO.15);
R3:5’-taactccctgtgattcc-3’(SEQ ID NO.16);
SEQ ID NO.4片段的引物为:
F4:5’-atgaggggtgaggaaggccg-3’(SEQ ID NO.17);
R4:5’-gtcatgcagtttttttatctg-3’(SEQ ID NO.18);
SEQ ID NO.5片段的引物为:
F5:5’-atgctggggccgcacatgttgc-3’(SEQ ID NO.19);
R5:5’-agatccagtcttggagc-3’(SEQ ID NO.20);
SEQ ID NO.6片段的引物为:
F6:5’-atgtccaaacaggagcttgatg-3’(SEQ ID NO.21);
R6:5’-aacaccaattctagtaaggac-3’(SEQ ID NO.22);
SEQ ID NO.7片段的引物为
F7:5’-atgtctttgattcgcaag-3’(SEQ ID NO.23);
R7:5’-gaaacgttgtttaatatttttc-3’(SEQ ID NO.24);
SEQ ID NO.8片段的引物为
F8:5’-atgtttaacattgcagatgg-3’(SEQ ID NO.25);
R8:5’-ctgctgctgggctacctgc-3’(SEQ ID NO.26);
以编码全长Mi-2抗原蛋白的DNA为模板,以上述8组引物分别进行PCR反应,得到8段目标片段。
PCR反应条件为:预变性:98℃,2min;变形、退火、延伸:98℃,10s;60℃,10s;72℃,20s;循环30次;保温:72℃,4min。
上述PCR产物分别经琼脂糖凝胶电泳、切胶回收、纯化得到编码SEQ ID NO.1-8的核酸片段。
【2】分别将【1】中的8段PCR产物***到已改造的pET-28a载体中(所述改造载体在pET-28a载体的酶切位点NcoⅠ和BamHⅠ间***MBPtag,并加入TEV酶切位点),PCR产物的***酶切位点为BamHⅠ和HindⅢ。
【3】分别将【2】中的8种重组质粒转化入E.coli DH5α感受态细胞中,并于LB琼脂培养平板上37℃倒置培养过夜,挑菌进行菌落PCR验证和测序,测序正确后扩大培养并提取质粒。
【4】分别将上述测序正确的质粒转化入E.coli Rosseta(DE3)感受态细胞中,并于LB液体培养基中37℃培养至OD600nm=0.6-0.8,加入IPTG至终浓度1mM,25℃下诱导培养4h后收集菌体。
蛋白纯化
【1】取出收集的菌体,加入裂解Buffer(裂解Buffer配方为:20mM Hepes,1M NaCl,5%Glycerol,pH8.0),冰上重悬菌体。
【2】超声破碎:按照超声功率400W,超声4S,停3S,循环200次的条件破碎细菌,直至菌液清亮透明为止。
【3】超声结束后,于4℃,12000rpm,离心30min(离心机提前预冷)分离上清与沉淀,上清使用0.22μM膜过滤。
【4】取10ml MBP resin用裂解Buffer平衡柱子5CV,然后加入过滤后的上清液中,混匀,置于冰盒中摇床振荡孵育40min-60min。
【5】将孵育好的混合物倒入重力柱,用干净的离心管收集流穿液,待流穿液全部流完之后,加入10CV的裂解Buffer将管路中残留洗净。
【6】洗脱:用20ml洗脱Buffer(洗脱Buffer配方为:20mM Hepes,50mM NaCl,5%Glycerol,10mM Maltose,pH8.0)进行洗脱并收集洗脱液,然后G25脱盐。
【7】酶切:按质量比1:20加入TEV酶,4℃下过夜酶切16h。
【8】酶切后溶液再次加入MBP resin,孵育后倒入重力柱,收集流穿液即为目的蛋白。
【9】蛋白电泳验证洗脱液中的目的蛋白纯度,纯度≥90%可用于抗原的验证。
液相芯片包被及反应性测试
将制备的各蛋白包被在固相载体上。
步骤1、活化缓冲液的配制
取2-(N-吗啉代)乙烷磺酸钠盐(C6H12NO4·SNa)21.7g,加入适量超纯水中,待完全溶解后,在25±1℃条件下调溶液pH至6.0±0.2,加入0.5ml的Proclin300,混合均匀,定容至1000mL。
步骤2、包被液的配制
取磷酸二氢钾(KH2PO4)0.24g、磷酸氢二钠(Na2HPO4·12H2O分子量357.96)2.89g、NaCl 8g、KCl 0.2g,加入适量超纯水中,待完全溶解后,在25±1℃条件下调溶液pH至7.4±0.2,加入0.5ml的Proclin300,混合均匀,加入定容至1000mL。
步骤3、液相芯片BMB的包被
固相载体为Applied BioCode公司的数码液相芯片(Digital Liquid Chip),其具有超顺磁性、且由生物兼容的高分子聚合物组成(Barcoded Magnetic Beads,BMB),每一种芯片具有独一无二的12位二进制的数字编码。BMB表面共价交联有丰富的羧基。
包被流程如下:
吸取所需体积的BMB混悬液;
活化缓冲液洗涤数遍;
加入一定量的EDC(1-乙基-3-[3-二甲基氨基丙基]碳化二亚胺盐酸化物)/NHS(N-羟基琥珀酰亚胺中文名称),室温振荡反应50mim,活化羧基;
包被液洗涤数遍;
加入制备好的抗原,室温振荡孵育60min;
封闭液(含1%BSA牛血清白蛋白、0.1%Tween 20的包被液)封闭BMB 60min;
含1%BSA牛血清白蛋白的包被液重悬。
4、荧光标记物
藻红蛋白标记的羊抗人IgG-PE:浓度为0.5mg/ml,用包被稀释液稀释至工作浓度0.5ug/ml。
5、反应流程
每孔加稀释好的样本到50μl BMB中,37℃孵育15min;
磁力洗板机洗3次,吸走残留液体;
每孔加入50μl荧光标记物羊抗人IgG-PE,37℃孵育15min;
磁力洗板机洗3次,吸走残留液体;
每孔加入100μl检测液,用50μl枪吸打混匀,消泡,静置30s检测。
试验例
1.筛选与阳性样本结合区段
分别对实施例1中所表达的8种蛋白和通过编码全长Mi-2抗原蛋白的DNA表达的全长Mi-2抗原蛋白进行验证。
先将待验证抗原包被液相芯片,然后加入已知阴性或阳性的血清样本反应,封闭洗涤后,最后用带有荧光素藻红蛋白(PE)的二抗孵育,再次洗涤后在荧光判读仪下检测对应的荧光值,根据荧光值的大小判定其阴阳性。
分别将以上所表达的抗原做验证,以全长Mi-2抗原蛋白作对照。验证实验选取53个阳性样本和45个阴性样本,分别验证各蛋白的阳性检出率即为灵敏度,阴性检出率即为特异性。
从以上检测结果可以看出:SEQ ID NO.1、SEQ ID NO.5和SEQ ID NO.7的灵敏度较全长Mi-2抗原均有较大提高,SEQ ID NO.1、SEQ ID NO.5和SEQ ID NO.7,这3个片段包含主要的抗原位点。为方便描述,命名SEQ ID NO.1为N,命名SEQ ID NO.5为M,命名SEQ ID NO.7为C。通过修改蛋白序列,使得抗原位点暴露的更加充分,使得对抗Mi-2抗体的特异性、灵敏度有所增强。
2.筛选与阴性样本结合区段
以全长Mi-2抗原蛋白为基础,分别构建8种重组蛋白(重组蛋白a-h),这8种重组蛋白相对全长Mi-2抗原蛋白分别缺少SEQ ID NO.1-8序列,然后分别对所表达的8种重组蛋白(重组蛋白a至h)和全长Mi-2抗原进行验证。
先将待验证抗原包被液相芯片,然后加入已知阴性或阳性的血清样本反应,封闭洗涤后,最后用带有荧光素藻红蛋白(PE)的二抗孵育,再次洗涤后在荧光判读仪下检测对应的荧光值,根据荧光值的大小判定其阴阳性。
分别将以上所表达的抗原做验证,以全长Mi-2作对照。验证实验选取53个阳性样本和45个阴性样本,分别验证各蛋白的阳性检出率即为灵敏度,阴性检出率即为特异性。
从以上检测结果可以看出:
相比于全长Mi-2抗原,在分别剔除SEQ ID NO.6、SEQ ID NO.5、SEQ ID NO.2后,重组蛋白f、重组蛋白e、重组蛋白b的灵敏度和特异性均较全长Mi-2抗原有提升。由数据分析可得,SEQ ID NO.6、SEQ ID NO.5、SEQ ID NO.2上具有易于使全长Mi-2抗原于阴性样本结合的区间结构,并且由于序列长度相比于全长Mi-2抗原有所减小,减少了与抗Mi-2抗体的空间位阻,因此特异性有所提升。
相比于全长Mi-2抗原,在分别剔除SEQ ID NO.4、SEQ ID NO.8时,重组蛋白d、重组蛋白h的灵敏度、特异性相较全长Mi-2抗原均无较大变化,因此推测,SEQ ID NO.4、SEQ IDNO.8的蛋白结构对识别结合抗Mi-2抗体、结合阴性样本均无较大作用。
相比于全长Mi-2抗原,剔除SEQ ID NO.3的重组蛋白c灵敏度有所提升,但特异性有所下降,推测SEQ ID NO.3的蛋白结构可利于与阴性样本结合。
结合SEQ ID NO.1、SEQ ID NO.5、SEQ ID NO.7的特异性及灵敏度结果,可推测SEQID NO.1、SEQ ID NO.7上包含主要的抗原位点,且无有利于阴性样本的结合位点,SEQ IDNO.5上即包含主要的抗原位点,同时也具有利于阴性样本的结合位点。为方便描述,命名SEQ ID NO.1为N,命名SEQ ID NO.5为M,命名SEQ ID NO.7为C。
3.融合蛋白的表达与验证
为增强对抗Mi-2的特异性及灵敏度,在尽量缩小抗原序列长度、减少结合时的空间位阻并更充分暴露抗原位点的前提下,把N、M、C这3个片段两两之间加入linker(GGGGSGGGG)拼接在一起,构建6个新的融和蛋白,分别是:融和蛋白NM(结构为SEQ IDNO.1-GGGGSGGGG-SEQ ID NO.5)、融和蛋白NC(结构为SEQ ID NO.1-GGGGSGGGG-SEQ IDNO.7)、融和蛋白MC(结构为SEQ ID NO.5-GGGGSGGGG-SEQ ID NO.7)、融和蛋白MN(结构为SEQ ID NO.5-GGGGSGGGG-SEQ ID NO.1)、融和蛋白CN(结构为SEQ ID NO.7-GGGGSGGGG-SEQID NO.1)、融和蛋白CM(结构为SEQ ID NO.7-GGGGSGGGG-SEQ ID NO.5)。
参照上述方案分别构建6个融和蛋白相应的表达质粒,在片段之间加入linker:GGGGSGGGG。参照上述试验过程,在E.coli中进行蛋白表达,分别进行纯化,再分别对此6种融和蛋白的抗原灵敏度和特异性进行验证,验证结果如下:
从以上检测结果可以看出:融合蛋白NC的灵敏度和特异性最好,较N、C蛋白均有较大提升。NM、MN、MC、CM的特异性远低于NC、CN是因为M上具有利于阴性样本的结合位点。
参照上述方案,把NC蛋白与M蛋白融合表达,分别构建2个表达质粒:MNC(SEQ IDNO.9)和NCM(SEQ ID NO.10)表达质粒,在片段之间加入linker:GGGGSGGGG。参照上述试验过程,在E.coli中进行蛋白表达,分别进行纯化,其中,融合蛋白NCM的SDS-PAGE检测结果如图1所示,再分别对此2种融和蛋白的抗原灵敏度和特异性进行验证,验证结果如下:
从以上检测结果可以看出:融合蛋白NCM,相对于Mi-2,减少了与抗Mi-2抗体结合时的空间位阻,提升对抗原位点的暴露,对抗Mi-2抗体的灵敏度有较大提高,由28.3%提升到90.6%(提升超3倍),特异性也由53.3%达到了88.9%(提升超1.5倍),抗原的性能满足检测试剂的需要,较NC蛋白也有所提升,因此重组融合蛋白NCM可替代Mi-2用作检测试剂抗原。
同时,NCM的灵敏度、特异性均优于MNC的原因,可能是因为M上的抗原位点位于序列后端,利于阴性样本的结合位点位于序列前端。NCM相较于MNC,将抗原位点暴露的更加充分的同时,增加了与阴性样本结合时的空间位阻,提升了NCM的灵敏度、特异性。
SEQ ID NO.4、SEQ ID NO.8上的蛋白结构对识别结合抗Mi-2抗体、结合阴性样本均无较大作用,SEQ ID NO.6、SEQ ID NO.5、SEQ ID NO.3、SEQ ID NO.2上具有易于使全长Mi-2抗原于阴性样本结合的区间结构。为增强对抗Mi-2的特异性及灵敏度,在尽量缩小Mi-2抗原序列长度、减少与抗Mi-2抗体结合时的空间位阻并充分暴露抗原位点的前提下,NCM为最佳Mi-2抗原序列。
尽管已用具体实施例来说明和描述了本发明,然而应意识到,在不背离本发明的精神和范围的情况下可以作出许多其它的更改和修改。因此,这意味着在所附权利要求中包括属于本发明范围内的所有这些变化和修改。
SEQUENCE LISTING
<110> 珠海丽珠试剂股份有限公司
<120> Mi-2重组抗原及其制备方法和应用
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Glu Arg Ile Thr Gln Val Ala Lys Lys Lys Met Met Leu Thr His Leu
690 695 700
Val Val Arg Pro Gly Leu Gly Ser Lys Thr Gly Ser
705 710 715
<210> 11
<211> 18
<212> DNA
<213> 人工序列
<400> 11
atggcgtcgg gcctgggc 18
<210> 12
<211> 18
<212> DNA
<213> 人工序列
<400> 12
gctctccacc acagctac 18
<210> 13
<211> 22
<212> DNA
<213> 人工序列
<400> 13
atggtgacag ccactgaggt tg 22
<210> 14
<211> 22
<212> DNA
<213> 人工序列
<400> 14
atggtggtca tcctcctctt cg 22
<210> 15
<211> 25
<212> DNA
<213> 人工序列
<400> 15
atggaattct gtcgggtctg caagg 25
<210> 16
<211> 17
<212> DNA
<213> 人工序列
<400> 16
taactccctg tgattcc 17
<210> 17
<211> 20
<212> DNA
<213> 人工序列
<400> 17
atgaggggtg aggaaggccg 20
<210> 18
<211> 21
<212> DNA
<213> 人工序列
<400> 18
gtcatgcagt ttttttatct g 21
<210> 19
<211> 22
<212> DNA
<213> 人工序列
<400> 19
atgctggggc cgcacatgtt gc 22
<210> 20
<211> 17
<212> DNA
<213> 人工序列
<400> 20
agatccagtc ttggagc 17
<210> 21
<211> 22
<212> DNA
<213> 人工序列
<400> 21
atgtccaaac aggagcttga tg 22
<210> 22
<211> 21
<212> DNA
<213> 人工序列
<400> 22
aacaccaatt ctagtaagga c 21
<210> 23
<211> 18
<212> DNA
<213> 人工序列
<400> 23
atgtctttga ttcgcaag 18
<210> 24
<211> 22
<212> DNA
<213> 人工序列
<400> 24
gaaacgttgt ttaatatttt tc 22
<210> 25
<211> 20
<212> DNA
<213> 人工序列
<400> 25
atgtttaaca ttgcagatgg 20
<210> 26
<211> 19
<212> DNA
<213> 人工序列
<400> 26
ctgctgctgg gctacctgc 19
Claims (7)
1.一种Mi-2重组抗原,其特征在于,所述Mi-2重组抗原为SEQ ID NO.9或SEQ IDNO.10。
2.与权利要求1所述的Mi-2重组抗原相关的生物材料,其特征在于,所述生物材料为如下任一种:
(a)核酸分子,编码权利要求1所述的Mi-2重组抗原;
(b)表达盒,含有(a)中核酸分子;
(c)重组载体,含有(a)中核酸分子或(b)中表达盒;
(d)重组真核细胞,含有(a)中核酸分子、(b)中表达盒或(c)中重组载体;
(e)重组原核细胞,含有(a)中核酸分子、(b)中表达盒或(c)中重组载体。
3.权利要求1所述的Mi-2重组抗原的制备方法,其特征在于,将编码Mi-2重组抗原的核酸分子导入宿主获得重组表达***,经表达得到Mi-2重组抗原。
4.根据权利要求3所述的制备方法,其特征在于,编码Mi-2重组抗原的核酸分子与pET-28a载体构建重组载体,将重组载体导入大肠杆菌,经表达得到Mi-2重组抗原。
5.权利要求1所述的Mi-2重组抗原在制备检测Mi-2抗体的产品中的应用。
6.权利要求1所述的Mi-2重组抗原在制备检测多发性肌炎的产品中的应用。
7.一种检测多发性肌炎的产品,其特征在于,所述产品采用权利要求1所述的Mi-2重组抗原作为检测抗原。
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Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6160107A (en) * | 1990-09-07 | 2000-12-12 | Oklahoma Medical Research Foundation | Nucleic acids encoding antigens associated with polymyositis and dermatomositis |
US6329517B1 (en) * | 1995-03-15 | 2001-12-11 | PRIVATES INSTITUT FüR IMONOLOGIE UND MOLEKULARGENETIK GMBH | Dermatomyositis-specific auto-antigen |
CN102138072A (zh) * | 2008-09-01 | 2011-07-27 | 学校法人庆应义塾 | 皮肌炎的诊断方法和诊断试剂盒 |
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2020
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Patent Citations (3)
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US6160107A (en) * | 1990-09-07 | 2000-12-12 | Oklahoma Medical Research Foundation | Nucleic acids encoding antigens associated with polymyositis and dermatomositis |
US6329517B1 (en) * | 1995-03-15 | 2001-12-11 | PRIVATES INSTITUT FüR IMONOLOGIE UND MOLEKULARGENETIK GMBH | Dermatomyositis-specific auto-antigen |
CN102138072A (zh) * | 2008-09-01 | 2011-07-27 | 学校法人庆应义塾 | 皮肌炎的诊断方法和诊断试剂盒 |
Non-Patent Citations (3)
Title |
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Autoantibody profiles in the sera of European patients with myositis;R Brouwera等;《Ann Rheum Dis》;20011231;图1、第117页右栏最后一段至第118页左栏第1段、摘要 * |
Molecular analysis of a major antigenic region of the 240-kD protein of Mi-2 autoantigen;Ge Q等;《The Journal of Clinical Investigation》;19951001;第96卷(第4期);1730-1737 * |
UniProtKB/Swiss-Prot: Q14839.2;NCBI;《NCBI》;20200812;ORIGIN * |
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Application publication date: 20201030 Assignee: Zhuhai Lihe medical diagnosis Products Co.,Ltd. Assignor: ZHUHAI LIVZON DIAGNOSTICS Inc. Contract record no.: X2024980010773 Denomination of invention: Mi-2 recombinant antigen and its preparation method and application Granted publication date: 20220125 License type: Common License Record date: 20240729 |